Neurons communicate by establishing connections, or synapses, with specific partners. Mechanisms that control the fidelity of these choices are poorly defined but are likely to depend on specific gene regulation. In the nematode, C. elegans, we have shown that the UNC-4 homeoprotein and transcriptional co-repressor, UNC-37/Groucho, function in VA motor neurons to block inputs normally restricted to sister VB motor neurons. We developed cell specific microarray profiling methods to identify UNC-4 regulated genes in this pathway. One of these targets, CEH-12, is homologous to the conserved homeodomain transcription factor, HB9, a known determinant of motor neuron fate in mammals. We used a powerful new strategy for visualizing motor neuron inputs to confirm that CEH-12/HB9 is in fact a VB gene normally repressed by UNC-4 to prevent the adoption VB-type inputs. These experiments also showed that this CEH-12 function is restricted to VA motor neurons in the posterior nerve cord and therefore suggest that UNC-4 must regulate other downstream genes to control synaptic inputs to anterior VAs. RNAi and genetic tests of additional candidate UNC-4 target genes from our microarray data sets have revealed that UNC-4 also negatively regulates the Frizzled protein and Wnt receptor, MOM-5. We hypothesize that a posterior source of Wnt signal acts through MOM-5 to drive ectopic expression of CEH-12 and VA miswiring in unc-4 mutants. A major goal of this project is to test this model by defining the molecular components of this Wnt signaling cascade and its mechanism of action. The existance of A/P gradients of Wnt signaling in the vertebrate spinal cord could be indicative of a similar Wnt dependent mechanism for specifying A/P differences in motor circuit connectivity. To identify unc-4 target genes that function in anterior VA motor neurons, we used high throughput genetic screens to isolate new mutations that "suppress" the Unc-4 movement defect. VA inputs in these mutants will be assessed with GFP labeled gap junction and synaptic proteins to confirm unc-4 pathway function. The DNA sequences of these UNC- 4 targets could be used in the future to identify similar genes in mammals where their roles in synaptic specificity can be explored. Thus, our work with a simple model organism is likely to provide important clues to fundamental processes governing the development of complex neural circuits in the the human spinal cord. PUBLIC HEALTH RELEVANCE Nerve cells (neurons) project elongated processes (axons) from the brain into the spinal cord to make connections or synapses with motor neurons that drive body movements. To facilitate the identification of genes that control the specificity of these connections, we are using the nematode, C. elegans, a model organism with a simple, well-defined nervous system. The results of this work should reveal similar human genes with crucial roles in the creation spinal cord circuits and therefore potentially contribute to the development of treatments for spinal cord injury and dysfunction. [unreadable] [unreadable]